Lyme disease, due to infection with the tick-borne spirochete Borrelia burgdorferi, is the most common vector-borne disease in the United States. After deposition in the skin during tick feeding, spirochetes disseminate widely and incite inflammation in several sites, especially the skin, heart, joints and nervous system. Using the murine model of Lyme borreliosis, much has been learned over the past two decades about the immune components that coordinate host defense against B. burgdorferi infection. In the initial stages of infection, phagocytes and T-cell independent antibodies are essential for the control of pathogen burden, but at the expense of producing acute inflammation and disease. During the adaptive phase, T and B cell responses are required for disease resolution. Saliva of the tick vector has pharmacologic properties that modulate these responses by inhibiting hemostasis and the function of phagocytes and T cells, thereby promoting B. burgdorferi survival within the host. These advances in understanding immune events provide only a mirrored view of B. burgdorferi maneuvering within the mammalian host. In this regard, relatively little is known about how spirochetes establish infection in the host or disseminate from the tick bite site. Similarly, the spatiotemporal coordination of immune responses to vector-borne Lyme borreliosis has not been defined. This proposal seeks to determine the feasibility of using multiphoton microscopy to visualize in a time-resolved fashion the initial stages of vector-borne B. burgdorferi infection. In Aim 1, we will use widefield and multiphoton microscopy to optimize conditions for static visualization of tick-transmitted spirochetes expressing green fluorescent protein (GFP) as they infect and begin to disseminate within the murine host. After determining optimal time points for analysis, skin of living mice infested with nymphs infected with GFPexpressing B. burgdorferi will be imaged by intravital microscopy to track the replication of B. burgdorferi, the kinetics of spirochete movement from the inoculation site, and spirochete interaction with skin architectural elements, including the vasculature. These studies will provide the first spatiotemporal analysis of hostpathogen interactions in a vector-borne infection. For the field of Lyme disease, completion of these studies will establish techniques for examining the in vivo maneuverings of B. burgdorferi and its counter moves to an evolving immune response and to antimicrobial therapy used to treat the human condition.